Production and characterization of a new diamine oxidase from Yarrowia lipolytica

Highlights

• A new microbial diamine oxidase was discovered in Yarrowia lipolytica.

• The gene was integrated into the genome of Y. lipolytica PO1f using CRISPR-Cas9.

• The enzyme was produced in a bioreactor by the modified Y. lipolytica PO1f.

• The enzyme had a broad substrate selectivity for highly relevant biogenic amines.

• This DAO shows promising characteristics for the food industry.

Abstract

A putative diamine oxidase (DAO) from Yarrowia lipolytica PO1f (DAO-1) was homologously recombinantly integrated into the genome of Y. lipolytica PO1f using the CRISPR-Cas9 system for the subsequent DAO production in a bioreactor. Thereby, it was proven that the DAO-1 produced was indeed a functional DAO. The cultivation yielded 2343 ± 98 nkat/L_culture with a specific DAO activity of 1301 ± 54.2 nkat/g_protein, which was a 93-fold increase of specific DAO activity compared to the native Y. lipolytica PO1f DAO-1 production. The DAO-1 showed a broad substrate selectivity with tyramine, histamine, putrescine and cadaverine being the most favored substrates. It was most active at 40 °C, pH 7.2 in Tris-HCl buffer (50 mM) (with histamine as substrate), which is comparable to human and porcine DAOs. The affinity of DAO-1 towards histamine was lower compared to mammalian DAOs (K_m = 2.3 ± 0.2 mM). Nevertheless, DAO-1 degraded around 75% of the histamine used in a bioconversion experiment with a food-relevant concentration of 150 mg/L. With its broad selectivity for the most relevant biogenic amines in foods, DAO-1 from Y. lipolytica PO1f is an interesting enzyme for application in the food industry for the degradation of biogenic amines.

Introduction

The biogenic amine histamine is especially found in foods that undergo a fermentation process due to the presence of microorganisms producing L-histidine decarboxylase (Santos, 1996). L-histidine decarboxylase (EC 4.1.1.22) generates histamine through the decarboxylation of the precursor L-histidine during the fermentation or storage of the food. Histamine exhibits a multitude of physiological functions in the human, acting as an important hormone and neurotransmitter (Timmerman and van der Goot, 2009). Therefore, the consumption of large amounts of exogenous histamine in food can cause serious poisoning with various physiological symptoms, such as vomiting, diarrhea or hypotension (Taylor et al., 1989). Other biogenic amines, such as tyramine, putrescine or cadaverine, are also frequently found in foods and can also cause toxicological effects in the human body (del Rio et al., 2019, Ladero et al., 2010). The consumption of moderate or even small amounts of histamine can also cause adverse allergy-like reactions in some susceptible individuals (Maintz and Novak, 2007). It is estimated that around 1% of the total population is intolerant towards histamine and, thereby, susceptible to minor dosages (Jarisch, 2013). This intolerance seems to derive from a disbalance between the amount of histamine ingested and the activity of the histamine degrading enzyme (EC 1.4.3.22) available in the small intestine (Maintz and Novak, 2007). This enzyme degrades histamine by oxidative deamination, resulting in the reaction products (imidazol-4-yl)acetaldehyde, hydrogen peroxide and ammonia (Schwelberger and Bodner, 1997).

There is no real treatment currently for the intolerance against histamine. People who are affected can administer a dietary supplement containing porcine DAO that is supposed to support the endogenous DAO in the small intestine (Izquierdo-Casas et al., 2019, Komericki et al., 2011, Schnedl et al., 2019, Yacoub et al., 2018). However, it has been shown that the activity required for a satisfactory histamine reduction is considerably larger than expected and that an alternative to the porcine DAO formulation used currently has to be found (Kettner et al., 2020). The diversity of enzymes found in microorganisms could have the potential of providing a competitive DAO with similar or even better attributes. If administered in a dietary supplement or generally in the food industry, an advance of microbial DAOs is the cost-effective and convenient enzyme production. A promising producer of an alternative microbial DAO could be Yarrowia lipolytica (Y. lipolytica) (Nuñez and Medina, 2011). This yeast represents one of the yeast species most frequently found in raw milk and is also found in various types of cheese (Fröhlich-Wyder et al., 2019). It contributes to the development of flavor and aroma during the ripening of the cheese due to its proteolytic and lipolytic activity and is, therefore, a desirable and important constituent of the product (Buehler et al., 2017, Jacques and Casaregola, 2008). The yeast occurs naturally in cheese since contamination can happen at different production stages (raw milk, air, brine, contact with surfaces) and it grows well under the common environmental conditions in foods (Atanassova et al., 2016). However, Y. lipolytica was classified as an opportunistic pathogen in 2018 by the European Food Safety Authority and was given the qualified presumption of safety status only for production purposes (EFSA et al., 2018). This means that the yeast should not be present in the final food as viable cells but is allowed to be used as an inactivated biomass as a novel food (EFSA et al., 2019). Y. lipolytica was identified as a biogenic amine producer but was also suspected of being capable of degrading biogenic amines during the ripening of the food (Atanassova et al., 2016, Nuñez and Medina, 2011). However, the enzymes responsible for this degradation of biogenic amines have not yet been identified or described in literature.

The aim of our study was to investigate these putative biogenic amine-degrading enzymes from Y. lipolytica. In fact, two putative DAOs (Uniprot: DAO-1 (Q6CGT2) and DAO-2 (Q6CB09)) were identified when using the BLAST program (https://blast.ncbi.nlm.nih.gov) to align the amino acid sequence of the well-described porcine DAO (UniProt: Q9TRC7) with the proteome of Y. lipolytica. To ensure correct production and folding of the putative DAOs, the genes for both were cloned homologously into the natural host Y. lipolytica PO1f strain. The gene products were investigated for DAO activity and biochemically characterized to assess their potential suitability for application in the food industry.